1. Field of the Invention
The present invention relates generally to nucleic acid probes useful in the detection and analysis of target nucleic acid sequences. More particularly, the present invention concerns nucleic acid probes wherein naturally occurring nucleobases or other nucleobase-binding moieties are covalently bound to an oxocarbonamide containing peptide backbone. In certain aspects, the present invention concerns methods employing nucleic acid probes in the detection and analysis of target nucleic acid sequences including, for example, mRNAs, miRNAs, and siRNAs.
2. Description of Related Art
A large number of small, non-coding RNAs have been identified and designated as microRNAs (miRNAs) (Ke et al., 2003). miRNAs have been shown to regulate gene expression at many levels, representing a novel gene regulatory mechanism. Understanding this RNA-based regulation will be useful to understand the complexity of the genome in higher eukaryotes as well as understand the complex gene regulatory networks.
miRNAs are 18-25 nucleotide (nt) RNAs that are processed from longer endogenous hairpin transcripts by the enzymes Dicer and Argonaute (Ambros et al., 2003; Grishok et al., 2001). To date more than 4160 microRNAs have been identified in mammals, birds, fish, worms, flies, plants, and viruses according to the miRNA registry database release 9.0 in October 2006, hosted by Sanger Institute, UK. Some miRNAs have multiple loci in the genome (Reinhart et al., 2002) and may be arranged in tandem clusters (Lagos-Quintana et al., 2001).
The first miRNAs to be discovered, lin-4 and let-7, base-pair incompletely to repeated elements in the 3′ untranslated regions (UTRs) of other heterochrony genes, and regulate the translation directly and negatively by antisense RNA-RNA interaction (Lee et al., 1993; Reinhart et al., 2000). Some miRNAs are thought to interact with target mRNAs by limited complementary and suppressed translation as well (Lagos-Quintana et al.,. 2001; Lee and Ambros, 2001). Perfect complementarity between miRNAs and their target RNA may lead to target RNA degradation rather than inhibit translation (Hutvagner and Zamore, 2002), which suggests that the degree of complementarity determines function.
Several human diseases have been identified in which miRNAs or their processing machinery might be implicated. One such disease is spinal muscular atrophy (SMA), a pediatric neurodegenerative disease caused by reduced protein levels or loss-of-function mutations of the survival of motor neurons (SMN) gene (Paushkin et al., 2002). Another disease linked to mi/siRNAs is fragile X mental retardation (FXMR) caused by absence or mutations of the fragile X mental retardation protein (FMRP) (Nelson et al., 2003). Poy et al. (2004) concluded that miR-375 is a regulator of insulin secretion and could constitute a novel pharmacological target for the treatment of diabetes. Links between cancer and miRNAs have also been described. For example, one study determined that two different miRNA (miR15 and miR16) genes are clustered and located within the deleted minimal region of the B-cell chronic lymphocytic leukemia (B-CLL) tumor suppressor locus, and both genes are deleted or down-regulated in the majority of CLL cases (Calin et al., 2002).
RNA interference (RNAi), in which double-stranded RNA leads to the degradation of any RNA that is homologous (Fire et al., 1998), relies on a mechanism that probably evolved for protection against viral attack and mobile genetic elements. One step in the RNAi mechanism is the generation of short interfering RNAs (siRNAs), double-stranded RNAs that are about 22 nt long. The siRNAs lead to the degradation of homologous target RNA and the production of more siRNAs against the same target RNA (Lipardi et al., 2001; Zhang et al., 2002; Nykanen et al., 2001).
The involvement of short RNAs in gene regulation has resulted in high interest among researchers in the discovery of siRNAs, miRNAs, their targets and mechanism of action. However, the detection and analysis of these small RNAs is not trivial. The size and often low level of expression of miRNAs require the use of sensitive analysis tools. The use of conventional quantitative real-time PCR for monitoring expression of mature miRNAs is excluded due to their small size. Most miRNA researchers use Northern blot analysis combined with polyacrylamide gels to examine expression of both the mature and pre-miRNAs (Reinhart et al., 2000; Lagos-Quintana et al., 2001; Lee and Ambros, 2001). Primer extension has also been used to detect the mature miRNA (Zeng and Cullen, 2003). Disadvantages of all the gel-based assays (Northern blotting, primer extension, RNase protection assays etc.) for monitoring miRNA expression include low throughput and poor sensitivity. Consequently, a large amount of total RNA per sample is required for gel-based methods, which is not feasible when the cell or tissue source is limited.
Microarrays are an alternative to Northern blot analysis for analyzing miRNA expression. Krichevsky et al. (2003) used cDNA microarrays to monitor the expression of miRNAs during neuronal development; however, the mature miRNAs had to be separated from the miRNA precursors using micro concentrators prior to microarray hybridization. Liu et al (2004) developed a microarray for expression profiling of 245 human and mouse miRNAs using 40-mer DNA oligonucleotide capture probes. Thomson et al. (2004) described the development of a oligonucleotide microarray platform for expression profiling of 124 mammalian miRNAs using oligonucleotide capture probes complementary to the mature microRNAs.
Although microarrays can provide high throughput, the disadvantages of DNA-based oligonucleotide arrays may include: the requirement of high concentrations of labeled input target RNA for efficient hybridization and signal generation, low sensitivity for rare and low-abundant miRNAs, and the necessity for post-array validation using more sensitive assays.
A PCR-based approach has also been used to determine the expression levels of mature miRNAs (Grad et al., 2003). However, this method is cumbersome for routine miRNA expression profiling, since it involves gel isolation of small RNAs and ligation to linker oligonucleotides. Schmittgen et al. (2004) described an alternative method to Northern blot analysis, in which real-time PCR assays were used to quantify the expression of miRNA precursors. The disadvantage of this method, however, is that it only allows quantification of the precursor miRNAs, which does not necessarily reflect the expression levels of mature miRNAs.
Many limitations of DNA probes for the detection of short nucleotide targets have been overcome by using locked nucleic acid (LNA) based probes or peptide nucleic acid (PNA) based probes. The use of LNAs and PNAs in oligonucleotide probes has been shown to increase sensitivity and selectivity for small RNA targets compared to their DNA-probe counterparts (see e.g., Valoczi et al., 2004). Nevertheless, additional compositions and methods are needed to increase the sensitivity and specificity of oligonucleotide sequences for the detection and analysis of miRNAs and other small RNAs, as well as for use in disease diagnostics and for antisense-based therapies.
The present invention addresses these needs by providing novel oligonucleotide compositions for the accurate, sensitive, and specific detection and functional analysis of miRNAs and other non-coding RNAs. The compositions of the present invention will also be useful as biomarkers for disease diagnostics as well as for antisense-based intervention targeted against disease-associated miRNAs and other non-coding RNAs.